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Tissue Printing and Dual Excitation Flow Cytometry for Oxidative Stress-New Tools for Reactive Oxygen Species Research in Seed Biology.

ABSTRACT: The intracellular homeostasis of reactive oxygen species (ROS) and especially of superoxide anion and hydrogen peroxide participate in signaling cascades which dictate developmental processes and reactions to stresses. ROS are also biological molecules that play important roles in seed dormancy and germination. Because of their rapid reactivity, short half-life and low concentration, ROS are difficult to measure directly with high accuracy and precision. In presented work tissue printing method with image analysis and dual excitation flow cytometry (FCM) were developed for rapid detection and localization of O₂^•- and H₂O₂ in different part of seed. Tissue printing and FCM detection of ROS showed that germination of wild oat seeds was associated with the accumulation of O₂^•- and H₂O₂ in embryo (coleorhiza, radicle and scutellum), aleurone layer and coat. To verify if printing and FCM signals were specified, the detection of O₂^•- and H₂O₂ in seeds incubated in presence of O₂^•- generation inhibitor (DPI) or H₂O₂ scavenger (CAT) were examined. All results were a high level of agreement among the level of ROS derived from presented procedures with the ones created from spectrophotometric measured data. In view of the data obtained, tissue printing with image analysis and FCM are recommended as a simple and fast methods, which could help researchers to detection and level determination of ROS in the external and inner parts of the seeds.

SUBMITTER: Cembrowska-Lech D

PROVIDER: S-EPMC7697308 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Tissue Printing and Dual Excitation Flow Cytometry for Oxidative Stress-New Tools for Reactive Oxygen Species Research in Seed Biology.

Cembrowska-Lech Danuta D

International journal of molecular sciences 20201117 22

The intracellular homeostasis of reactive oxygen species (ROS) and especially of superoxide anion and hydrogen peroxide participate in signaling cascades which dictate developmental processes and reactions to stresses. ROS are also biological molecules that play important roles in seed dormancy and germination. Because of their rapid reactivity, short half-life and low concentration, ROS are difficult to measure directly with high accuracy and precision. In presented work tissue printing method ...[more]

PMID: 33212814

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Project description:1. The fluorescent compound 2',7'-dichlorofluorescein was used as an indicator of intracellular H2O2 production by neutrophils in order to compare the response of the cell population with that observed with individual cells determined by flow cytometry and quantitative fluorescence microscopy. 2. 2',7'-Dichlorofluorescein diacetate was deacetylated by intracellular esterases to form reduced 2',7'-dichlorofluorescein. The polar non-fluorescent intermediate remained trapped within both intracellular granules and the cytoplasm. Reduced dichlorofluorescein was oxidized by H2O2, a product of the oxidative burst, to yield the highly fluorescent product dichlorofluorescein. 3. A population of neutrophils stimulated by suboptimal concentrations of fMet-Leu-Phe (N-formylmethionyl-leucyl-phenylalanine) or phorbol ester (phorbol 12-myristate 13-acetate) resulted in an oxidation of 45-50% of the cellular dichlorofluorescein (non-fluorescent) to oxidized dichlorofluorescein within 30 min. Subcellular fractionation showed that, although dichlorofluorescein (non-fluorescent) occurred both in the cytoplasm and the granules, oxidation of dichlorofluorescein (non-fluorescent) occurred predominantly in the granules of stimulated neutrophils. 4. Flow cytometry showed that unstimulated cells consisted of a single population of cells with low cellular fluorescence. Activation of neutrophils (to produce reactive oxygen metabolites) resulted in the appearance of a second population of cells, with high fluorescence. The number of cells in this new population increased with time. fMet-Leu-Phe (0.1 microM) or phorbol ester (1 ng/ml) activated 45% of the cells within 8 min and 42% within 30 min respectively. 5. Analysis of individual cells by quantitative fluorescence microscopy demonstrated that, in the presence of a suboptimal concentration of stimulus, cells either failed to respond or were activated after different time delays, 4-120 s (39 +/- 18.4 s) by fMet-Leu-Phe or 12-200 s (59 +/- 17.4 s) by phorbol ester. Furthermore the oxidative bursts were of different magnitudes. 6. It is concluded that, in order for an individual cell to cross the activation threshold for the 'end response', a critical concentration of stimulus together with the necessary changes in intracellular signals are required.

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Dataset Information

Tissue Printing and Dual Excitation Flow Cytometry for Oxidative Stress-New Tools for Reactive Oxygen Species Research in Seed Biology.

Publications

Tissue Printing and Dual Excitation Flow Cytometry for Oxidative Stress-New Tools for Reactive Oxygen Species Research in Seed Biology.

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